Midterm 1 Flashcards
1
Q
Hypothesis
A
statement consistent with most of the data might take the form of a model
explanation which seems to account for the data
2
Q
important thing about hyp
A
must be testable
3
Q
hyp is best when it is
A
1 possibility out of many
4
Q
theory
A
hypothesis that have been extensively tested by many investigators, using diff approaches and is widely accepted
decades of testing
5
Q
law
A
proven with the outcomes that are predictable and can be calculated
not absolute, can be refuted
6
Q
level of confidence in hypothesis , theory and law
A
little in hyp
lot in theory and law
7
Q
facts
A
tenuous and dynamic ( results of exp.ts)
8
Q
Occam’s razor
A
simplest explanation consistent with facts most likely correct
9
Q
complexity and hyp
A
as complexity increases, hyp hard to test
10
Q
three strands contributing to modern biology
A
cytology
biochemistry
genetics
11
Q
cytology
A
field with emphasis on optical techniques and cellular structures
12
Q
biochemistry
A
focuses on cell function
13
Q
genetics
A
info flow and heredity
14
Q
1665 cytology
A
Hooke microscopist, cork structure was observed
first named cells
achieved 30 times magnification
15
Q
Cytology timelie
Robert Hooke, Antonie Leeuwoek, Theodor Schwann, Rudolf Virchow
A
Hooke 1665 30X microscopy cells
Antonie 300X microscopy
Schwann 1839 Cell theory
Virchow 1855 preexisting
16
Q
Antonie Leuwenhook
A
300X microscopy
17
Q
early progress in cell biology was hindered by
A
limited resolution ( ability to see fine detail)
descriptive nature of biology
focus on observation not on explanation
18
Q
Theodor Schwann
A
1839
Cell theory
1. all org 1 or more cells
2. cell basic unit of struture for all org
19
Q
Rudolf Virchow
A
1855
Add to cell theory
3. All cells derive from pre existing cells
20
Q
competing theory with Virchow
A
spontaneous generation
leave meat out and maggots seem to come from nowhere to grow on it
21
Q
Biochemical strand timeline
Friedrich Wohler, Edward Buchner
A
Fredrich Wohler -1828
urea
Edward Buchner -1897 enzymes
22
Q
Fredrich Wohler
A
1828 synthesized urea in a lab
contradicted vitalism, the idea that living things were alive because of some special force
23
Q
Edward Buchner
A
1897
showed that sugar could be fermented using yeast extract
no living yeast present
enzyme brought about fermentation led to discovery of enzymes
24
Q
Genetic strand timeline
Gregor Mendel,Walter Flemming, Wilhelm Roux and August Weisman, Walter Suton and Boveri, Thomas morgan, calvin bridges and alfred strutevant, George beadle+Edward Tatum, James Watson+ Francis Crick
A
Gregor Mendel-1866 pea experiments
Walther Flemming1880 chromosomes
Wilhelm Roux and August Weisman suggested chrom carried genetic material
Walter Suton, Theodor Boveri - chromosome theory of heridity
Thomas morgan,calvin bridges,alfred strutevant
George Beadle + Edward Tatum
James Watson+Francis Crick
25
Cell biology timeline
Robert Hooke, Antonie Leeuwoek, Theodor Schwann, Rudolf Virchow, Friedrich Wohler, Edward Buchner, Gregor Mendel,Walter Flemming, Wilhelm Roux and August Weisman, Walter Suton and Boveri, Thomas morgan, calvin bridges and alfred strutevant, George beadle+Edward Tatum, James Watson+ Francis Crick
```
Hooke 1665 30X microscopy cells
Antonie 300X microscopy
Fredrich Wohler -1828 urea
Schwann 1839 Cell theory
Virchow 1855 preexisting
Gregor Mendel-1866 pea experiments
Walther Flemming1880 chromosomes
Wilhelm Roux 1883 and August Weisman suggested chrom carried genetic material
Edward Buchner -1897 enzymes
Walter Suton, Theodor Boveri 1900s - chromosome theory of heridity
Thomas morgan,calvin bridges,Alfred strutevant 1920s
George Beadle + Edward Tatum 1940s
James Watson+Francis Crick 1953
```
26
Gregor mendel
pea experiments laid foundation of understanding the passage of heridetary factors known as genes from parents to offspring
27
Lamarkian inheritance
competing hypothesis to Mendel's hypothesis that the experience of an organism is passed on to the offspring
28
Walter Flemming
1880
saw threadlike bodies in nucleus and called them chromosomes ( stained very stronngly with dyes) chromosomes another word for coloured bodies
named process of cell division mitosis
29
Wilhelm Roux and August Weisman
1883
| suggested chromosomes carried genetic material
30
Walter Sutton and Theodor BOveri
chromosome theory of heridetary
| proposed mendel's inheritance factors are located on chromosomes
31
Thomas Morgan, Calvin Bridges, Alfred Strutevant
connected specific traits to specific chromosomes on Drosophilla melanogaster ( common fruit fly)
32
chromosome theory
sex linked characteristics are inherited together
chrom carries discrete number of heriditary units
adopted gene from Wilhelm Johansen
concluded genes were possibly arrange in a linear fashion on chromosomes
33
George beadle and Edward Tatum
proposed the 1 gene 1 enzyme concept
| each gene produces one protein
34
James Watson + Francis Crick
DNA double helix structure
35
in vitro vs in vivio vs in sillico
in vitro outside of the cellular context reconstituted cellular activity
in vivo using live cells or organisms
in sillico model behaviors of cells and molecules and predict how they will be have
can be used to identify correlations
36
model organism key points
similar biology, less complex, reproduce quickly, cost, ethics
37
Common model organisms
```
E. coli
S. Cerevisiae ( yeast)
Drosophila ( fly)
Mus musculus ( mouse)
C. elegans
Arabidopsis
```
38
interesting fact about c. elegans
same number of cells in every organism
| used for developmental biology
39
Drosophila are used for
genetics
40
light micr allows identification of
organelles within cells
41
set up of light microscope
light source
lens gather as much light as possible and focus on the specimen
light has to go through the specimen
objective lens refocus the light and direct to eye
42
visible, UV and IR light ranges
vis: 400-700
UV less than 400
IR greater than 700
43
compound microscopes
2 lenses
increased magnification and resolution
1 micrometer samples could be seen
44
Robert Brown
used comp microscope
| identified nuclues in a cell
45
matthias schleiden
all plants were composed of cells
46
thomas schwann
all animals are composed of cells
47
microtome
allows for preperation of thin slices of sample
48
why use dyes
improve the limit of resolution ( how far apart objects must be in order to be considered distinct)
49
limit of resolution and resolution power
smaller limit of resolution indicates greater resolving power
50
resolutiion formula
0.612 wave/ NA
51
better resolution
smaller number
52
NA
refractive index of material you are looking at multiplies by the sin of the angle theta
increases as the lens increases in size
measure of
53
Best NA obtained with
oil which is 1.4
54
phase contrast and differential interference contrast
expoit differences in phase of light passing through a structure with a refractive index different than the surrounding medium
can be used to see living cells
55
phase contrast
converts phase shift in light passing through a transparent specimen to brightness variation
56
distinction between differential interference contrast (DIC) and phase contrast microscopy
hase contrast microscopy produces image intensity (amplitude) values that vary as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background. Alternatively, specimen features that have relatively low thickness values, or a refractive index less than the surrounding medium, are rendered much lighter when superimposed on the standard (positive) phase contrast medium gray background.
For differential interference contrast, optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for contrast. Steep gradients in path length generate excellent contrast, and images display the pseudo three-dimensional relief shading, which is characteristic of the DIC technique. Regions having very shallow optical path slopes, such as those observed in extended, flat specimens, produce insignificant contrast and often appear in the image at the same intensity level as the background.
57
differential interference
works by separating a polarized light source into two orthogonally polarized mutually coherent parts which are spatially displaced (sheared) at the sample plane, and recombined before observation. The interference of the two parts at recombination is sensitive to their optical path difference (i.e. the product of refractive index and geometric path length). Adding an adjustable offset phase determining the interference at zero optical path difference in the sample, the contrast is proportional to the path length gradient along the shear direction, giving the appearance of a three-dimensional physical relief corresponding to the variation of optical density of the sample, emphasising lines and edges though not providing a topographically accurate image.
58
Flourescence microscopy
detects labels or flourescent dyes to show locations of substances inside the cell
used to detect specific elements such as proteins
59
flourescet molecules
absorb one wavelength of light and mit another longer wavelength
60
confocal scanning
uses laser beam to illuminate a single plane of a flourescently labelled organism
61
flourescence micro set up
source with all wavelengths of light
monochromator selects for blue light or one lavelength
let only blu through
the illumination goes towards the eye
62
fluorescence is the only mode in optical microscopy
where the specimen produces own light
63
DAPI
dye which only stains nucleic acids
| especially AT rich regions in DNA
64
antibody
binds to a specific antigen
| prot binds to antigen, can be used to paint the protein to identify it
65
flourescence vs phase contrast
phase: allows to see the whole cell
flourescence: biased, only see a specific part
66
similarities widefield vs confocal microscopy
Both the confocal and the widefield microscope deliver fluorescent excitation light through the objective lens into the specimen.
Both the confocal and the widefield microscope have an resolution limited by the objective, NOT the instrument.
Both the confocal and the widefield microscope won’t avoid or break the laws of resolution limits, like super resolution systems do
67
wide vs confocal
widefield unable to see signal if it is perfectly in line
confocal improved z discrimination allows more accurate signal discrimination
focus on one slice of the specimen, allows to take whole bunch of images like deli cutter
elimination of out of focus light
68
specimens wide and confocal
can be living
69
labelling cells for flourescence microscopy
cells are harvested by centrifiguation
resuspended in some liquid
put on poly L Lysine slides
cells are fixed using formaldehyde and ethanol and membrane is permeabilised by poking holes into the cellular membrane, this kills the cell
protein is freeze fixed onto the membrane
slides incubated with blocking portein so that primary antibody does not bind to memrane
primary antibody binds to protein of interest
more common for primary to be unlabelled and then secondary antibody recognize primary antibody
1 or 2 secondary can recognize common proteins in all primary anti therefre less expensive
70
problem with flourescence
static, disruptive and limited by antibodies
71
green flourescent [rptein
can be used to see liing porteins
green under blue light
can put sequence into the cell and the organism with a e this proterty
same protein except that the protein is now flourescent, does not damage proteins
now there is a rainbow of proteins
72
superresolution
uses schotastic resolution of flourophores which blink on and off
if reapply gaussian distribution can reconstruct where the signals are seen
73
atomic force microscopy
analogous to braile
have an arm with a tip on it the size of an atom which gently scans the object back and force and takes a surface contour map wth neede
so small and fast that it can be used to scan processes such as myosin five walking on actin
74
electron microscope
limit of resolution 0.1-0.2 nm
sample has to be dead because have to coat sample with dense coat of gold
magnification up to 100000X much higher than light microscopes
uses electrons
75
TEM
electrons pass through the sample
used to study the ultra structure of cell and its components
structures as small as protein molecule or nano level can be use to see
based on transmitted electrons or produces images by detecting electrons transmitted from a sample
76
SEM
surface of specimen is scanned ,detecting electrons deflected from the outer surface
produces excellent images of surfaces of cells and organisms
excellent for studying surface morphology of organisms, cells or any suitable material
77
SEM vs TEM resolution and magnification
TEM has higher magnification and greater resolution
78
SEM vs TEM depth of field
SEM high TEm moderate
79
SEM vs TEM basis
SEM based on scattering electrons or detects secondary electrons which are emitted from the surface due to excitation by the primary electron beam
TEm based on transmission of electrons or detecting primary electrons from the sample
80
medium for SEM and TEM
high vacuum
81
SEM advantage over TEm
produces 3D images wherease TEM is only 2D
82
x- ray crystallography
make concentrated solution of DNA or protein and allow it to cool slowly which causes t to be more compact, atomic resolution diffraction pattern
83
important about C bonds
bonds are reversible
84
water and cell weight
makes up 70% of the cell weight
85
heat and water
heat which is released is first used to break numerous hydrogen bonds,
high specific heat capacity thus takes a lot of enrgy to increase temperature
thermal buffer
protects living systems from extreme temperature changes
86
repeating units in macromolecules
called monomers
87
examples of monomers
glucose, amino acids, nucleotides
| simple chemical structure
88
macromolecules
give life form and order and are generated by polymerization of smaller subunits
89
cellular heirarchy
biological moelcules and structures and organized into a series of levels which each build on the preceding one
90
supramolecular structures
made up of macromolecules
| macro can also function on their own
91
supramolecular structures which are components of
organelles and other subcellular structures which make up the cell
92
self assembly
the assembly of different pieces happen spontaneoulsy and automatically
process in which a disordered system of pre-existing components forms an organized structure or pattern as a consequence of specific, local interactions among the components themselves, without external direction
information needed to specify specific folding of macromolecules and interactions to form complex structures is inherent in the polymers themselves
93
structures consisting of complexes of 2 or more examples
cell membranes, ribosomes, mitochondria
94
advantages of heirarchal assembly
chemical simplicity: relatively few subunits used in variety of structures; allows small number discrete entities ito interact with each other and stabilize into a higher order structure and those to interact medium range with another similar group increasing complexity as a group
efficiency: cell never has enough enzymes to build all parts of molecules seperately, only have finite number of reactions it catalyzes, allows for efficiency small numbers and kinds of reactions are needed ( enzymes)
cells can make mistakes- when mistake occurs can pop out the mistake and put in a new one rather than totally replace everything
95
covalent bods and molecules
important in linking monomers of macromolecule together and stabilizing 3D structure
96
non covalent and molecules
important in folding of macromolecules
97
rank the following non covalent from strongest to weakest: ionic, hydrogen bond, van der waals, hydrophobic
hydrophobic- because so unfavourable to interact with polar solvent, very difficult to get apart
ionic- like in cooking an egg, impossible to et apart
HB- specialized type of ionnic except molecules do not carry a full charge, so it is weaker than ionic
van der waals close range
98
9 classes of proteins and their overall function
enzymes - catalysts which decrease activation energy for chemical reaction
structural- physical support and shape
motlity - contraction and movement
regulatory- control and coordinate cell function
transport- move substances in and out
hormonal- cellular communication
receptor- enable cells to respond to stimuli in the external environment
defensive- protect against disease and damage
storage- sinks for amino acids
99
hydrophobic vs hydrophillic and on protein
hydrophillic will be on surface of protein, charged ionic interactions to drive protein folding
hydrophobic in middle except pro
100
non polar amino acids
gly, ala, val, leu, ile , trp, met, phe, pro
101
polar uncharged aa
ser, tyr, thr, cys, asn, gln
102
polar charged aa
glu, asp, lys, arg, his
103
basic aa
arg, lys and his
104
acidic aa
glu, asp
105
protein synthesis
process of elongating chain of aa
106
polypep vs protein
polypepe becomes a protein when it has unique stable 3D shape and function
107
recombinant protein expression
take gene of interest , clone and place into a plasmid, inserted into bacteria with initiator sequence to turn on the gene
break bacteria open after protein is manufactured
identify protein and add a short tag sequence of His string
His tag binds to metal beads in collumn and are retained while the other proteins come through
add imidazole and the protein will come out all by itself
purified protein
108
gel electrophoresis contains
a jelly matrix which is made of gel or accrylamide
109
gel electrophoresis anode and cathode
cathode - charged is above
| anode + charged is below
110
SDS
ionic detergent which rips protein apart and gives it a negative charge so that it can be run in a gel electrophoresis
111
mercaptoethanol
pull apart the diS bonds of the protein so that they can run through collumn
112
speed moving through gel
smaller molecules move quicker
large molecules move more slowly
seperation based on size AND CHARGE
113
diff between DNA and RNA
DNA no 2' OH
| RNA 2' OH
114
Electrophoresis
group of techniques that use electric field to seperate molecules
115
seperation in electrophoresis
based on size and charge of molecule
116
support media of electro
agar comes as powder, cool to make matrix little molecules polyacrylamide comes as little molecules
117
DNA gel electrophoresis steps
make the gel
use ionic SDS to give negative charge if non before, no need to pretreat DNA
put in well, apply voltage
gels are horizontal so pipetting DNA in is easier
bigger fragments are slower
118
horiz vs vertical gel
horizontal typically agarose
| vertical typically polyacrylamide
119
temperature and gel movement
the hotter the gel is the faster the DNA fragments move, however if it s too hot it will melt
120
staining for DNA
ethidium bromide
| sticks to DNA and bases and glows in the dark when stuck to DNA
121
polysachs
long chains of sugar molecules whcih are not info molecules
122
storage polysaccharides
glycogen in animal cells and bacteria
starch in plants
alpha D glucose by alpha glycosidic bonds
123
cell size and shapes
take on diff shapes based on what they do some have little Er and others have a lot of ER
every cell has same DNA but not all same genes on at the same time
124
cell size limitations
need adequate surface area versus volume for exchange of gases but this ratio decreases with increasing size
rates at which molecules diffuse
maintain adequate local concentrations of substances required for necessary cellular functions
125
cells specialized for absorption
such as intestinal cells have microvilli which are finger like projections that increase SA
126
ways to avoid limitations porblem of low diffusion rates
```
using carrier proteins that transport materials thru the cytoplasm
cytoplasmic streaming ( cyclosis) to actively move cytoplasmic contents
move molecules using vesicle transport along microtubules
```
127
cytoplasmic streaming
movement of the fluid substance (cytoplasm) within a plant or animal cell. The motion transports nutrients, proteins, and organelles within cells.
creation of current inside cell
128
frequency of collisions and reaction
freq is increased by higher concentrations of enzymes and reactants, which increase as cell size increases
129
organlles
isolate enzymes and ions because diff chem reactions not all happen at the same time, represent compartmentalize of cell function
130
central dogma
refers to the flow on information from DNA to RNA to protin
131
protein and nucleic acid interconversion^
protein cannot go back to mRNA , info coded in protein is permanent which is not consistent with central dogma
132
refinements to central dogma
RNA can be the final product, RNA can be genetic material such as in viruses
133
functions of tRNA , mRNA and rRNA
tRNA: make protein
mRNA: template to make protein
rRNAL make ribo
134
RNA as genetic info
viral RNA makes DNA copy using reverse transcriptase
enzyme brings with it into the host
insert into host DNA and get viruses generated when it replicates
135
retrotransposons
short genetic elements which exist only to replicate themselves,
encode proteins which have 2 activities, endonuclease and reverse transcriptase
fragments of ancient viruses
RNA is still attached to the protein,
endonuclease cuts in the DNA
reverse transcriptase makes a DNA copy of the RNA on the cut DNA strand
host repair machinery fixes the lesion and replicates DNA to other cell
136
genetic code
relatioship between DNA base sequence and amino acids
| triplet code where 3 bases equals 1 aa
137
genetic code is
degenerate
| and non overlapping
138
Start codon
AUG
139
stop codons
UAA, UAG, UGA
| terminate synthesis
140
non overlapping code evidence
Brenner studied mutations in T 4 phage
addition or deletion
used ethidium bromide which slides between bases and causes addition or subtractions
found if lost or gained 1 or 2 had diff function than is lost or gained 3
141
frameshift mutations
shifting a reading frame by inserting or deleting a nt
142
overview of transcription
promoters recognized by transcription factors and mark where transcription takes place
RNAP binds to promoter, causes bubble by breaking HB between 2 DNA strand, whcih introduces local unwinding
5 prime to 3 prime polymerization using 1 strand
therefore when reading 3 prime to 5 prie on DNA , RNAP makes 5 prime to 3 prime
any mismatch on DNA will replicate one strand
moves along until reaches termination signal
143
how is transcription controlled?
by regulating access
144
Bacteria transcription and translation
bacterial transcription and translation are tightly coupled
145
detailed overview of transcription
promoters are sequence specific
TF 2D binds to TATA box, lots of variations of TATA
TF scans through the DNA until finds TATA and snaps shut
TF A and B come and form complex
open mouse trap which recognizes TF 2D when it is bound and snaps on
TF 2 F and RNAP2 can recognize complex once it contains TF
TF E and H recognize conformation and come on
this is the pre initiation complex
phosphorylation changes chemical event of side chain and RNAP 2 phsophoryl turns on and goes
146
do all RNAP require TF
yes
147
RNAP 1 , 2 and 3 and what they do
1: nucleolus rRNA
2: nucleoplasm mRNA, snRNA
3: nucleoplasm 5S rRNA , tRNA
148
primary transcript
newly produced RNA molecule after transcription
149
RNA processing
chemical modifictaion that all primary transcripts must go through before they can function in the cell
150
heterogenous nuclear RNA
hnRNA mixture of mRNA molecules and their precursors
151
RNA processing
modified nucleotide called 5 prime cap which is a methylated cap and a 3 prime poly A tail which consists of a long string of As
152
5 prime cap nucleotide modification
guanosine which is methylated at the position 7 of the purine ring
153
5 prime cap is bound to the RNA
by 5 prime to 5 prime bond
154
purpose of the 5 prime cap and the poly A tail
so the cell can identify the nucleic acid as its own and will not degrade it
155
role of teh 5 prime cap
added soon after transcription is initiated
increases mRNA stability by protecting the RNA from nucleases
plays a role on positioning RNA on ribosome for initiation of translation
156
poly A tail
ranges from 50 to 250 nts long
clevage and polyadenylation specificity factor cleaves 3 prime most prt of mRNA
polyadenylate polymerase adds the poly A tail by adding AMP to the 3 prime and cleaving off pyrophosphate
157
function of poly A tail
protects mRNa from nuclease attack
length of tail influences stability
required for export of transcript to the cytoplasm
help ribosomes recognize and bind mRNAs
158
introns
sections of the mRNA transcript that do not encode protein
need to be removed prior to translation
if an intron is not removed, it remains as part of the final RNA molecule. The translation of its sequence alters the sequence of the protein product, most often causing (a) frameshift with premature stop codons or (b) incorrect skipping of exon(s)
159
RNA splicing
process of removing introns and joining exons
160
5 prime and 3 prime splice sites are
determined by sequences commonly found at exon and intron boundaries
161
spliceosomes
massive prot nucleic acid complexes
consisting of 5 types of RNA and many proteins
snRNPS small nucleic ribo protein complexes are smaller RNA protein comlexes that these assemble from
162
snRNPs
contain each one or tow snRNAs
163
splicing and RNA synthesis
splicing is coupled with transcription
164
splicing mechanism overall
exon 1 and 2 have key sequences
GU at 5 prime end A near 3 prime end and EG at 3 prime end of exon
5 prime splice site recognized by U1 SnRNP
A is bound by U2 snRNP
U4/U6 and U5 binds
creates a lloop
ribonucleocomlexes are also enzymes, thus breaks 5 prime back bone upstream of GU and attaches to A in a lariat structure
RNA is cleaved at 3 prime site and a exon junction complex EJC is added when intron is left
165
EJC
happens to be everywhere where splicing occured
166
self splicing RNA introns
group 1 and 2
| RNAs which carry out splicing without the help of proteins
167
ribozymes
RNA molecules which also function as catalysts
168
why splicing
presence of introns allows for the mRNA to be sliced in multiple ways
leading to production of many diff protein products
alternative splicing have mechanisms allowing certain sites to be ignored
regulatory proteins and snoRNAs bind to splicing enhancer or silencing sequences
snoRNAs guide the splicing to sites
can lead to mRNAs with introns in it whcih can encode somtheing
169
why is mRNA turned over so quickly
Responding to changing conditions
Most responses to changing stimuli , particularly bacteria, they have to adapt very quickly going into salty water
If you do not have mRNAs that turn over that are useless hard to have mRNAs mean anything at a protein level
170
amplification of genetic info
mRNA can be synthesized many times from same piece of DNa and mRNA can be translated many times, leading to amplification of genetic info
171
missense mutation
changed the codon from coding one to a totally different one
172
silent mutation
don't alter the aa but the codon has changed
| Can cause problems with nearby binding sites recognition by snoRNAs
173
nonsense mutation
type of missense mutation
| convert amino acid coding coons into stop codons leaidng in incomplete, trunkated peptides
174
nonsense mediated decay
used to destroy mRNAs with a premature stop codon
175
nnonstop muattion
removal of stop codon can cause the translation to stall when ribo reaches end of the transcript
176
non stop mediated decay
RNA degrading enzyme binds to the stalled ribo and degrades the defective mRNA
177
nonsense mediated decay
sensed by the fact that both the EJC ( which should get ripped off as the ribosome translates) is on the thing and the termination complex which the ribo builds at a termination complex
triggers cascade that drives degradation
178
non stop mediated decay
non stop strip off all the EJCs and never make termination complex
Maybe it is ribo bumping in to PABPs
Maybe ribo stalls because it runs out of tRNAs for that amino acid
Something in the process triggers decay
179
rRNA
most abundant and stable form of RNA in cells
180
4 types of rRNA and subunit they correspond to
small: 18S rRNA
large: 28S , 5.8 S,5S
181
rRNA making process
A series of copies of a gene arranged in tandem along a chromosome
1 pre RNA is responsibel for 3 rRNAs
transcribed as a set
need same numbers and stoich ratio of the rRNAs ,
transcription by RNA polymerase I to make pre rRNA
RNA processing cleavage
transcribed spacers degraded
182
rRNA processing
Have base methylation, ribose methylation, pseudouridylation
modifications and cleavage of rRNA are guided by snoRNAs
183
pseudouridylation
Pseudouridylation base lopped off and connected
Reverses the polarity of the base
Reading backwards with respect to how it would bond
Part of it might be read parallel not antiparalel
184
nucleolus
non membrane organelles in the nucleus
185
Energy altering RNA
Efficiency, easy to fix something , keeps simple as possible get new base where we need it
Can be changed depending on where they are and what cells they are expressed in
186
S
sedimentation
187
centrifugation
rate of movement through a solution depends on its size and density
188
sedimentation rate
rate of movement through a solution
189
centrifuge
piece of equipment that consists of a rotor that can be spun rapidly in a circular motion by an electric motor
Allows you to sediment a solution if you have a cell homogenate with nuclei organelles, heaviest stuff will go to the bottom
Can take advantage of size and density differences
190
differential centrifugation
Free proteins small organelles stay in the supernatant , nucleus is large and slam to the bottom slow spin
Take supernatant spin a little higher organelles pellet out
Can do at diff speeds and isolate diff components according to their size
Can fractionate the components of the cell
Sedimentation coefficient how easy does it pellet, the bigger the number, the bigger it is
larger sedimentation coefficients pellet first
larger S is larger moelcule
Particles of different densities or sizes in a suspension will sediment at different rates, with the larger and denser particles sedimenting faster
191
sedimentation coeff
measure of how rapidly particle sediments when subject to centrifugation
192
sub cellular fractionation
using centrifugation to isolate and purify organelles and macromolecules based on sedimation rates and density
193
density centrifugation
a procedure for separating particles such as viruses or ribosomes or molecules such as DNA in which the sample is placed on a preformed gradient such as sucrose or cesium chloride. Upon centrifugation either by rate zonal or equilibrium procedures, the macromolecules are 'banded' in the gradient and can be collected as a pure fraction.
Diff densities of solution in the tube
Layer your cell mixture
Spin at a speed for long time , these components will float at their respective densities
194
monocistronic
one gene can only make one polypep
| euk
195
bacteria and archaea polycistronic
encode several peptides
| polycistronic units are called operons
196
ribosomes
carry ot the process of translation
197
mRNA
encodes amino acid sequence info
198
amino acyl tRNA synthetases
attach aa to approp tRNA molecules
199
tRNA
align aa in correct order
200
regulatory protein factors
facilitate initiation, elongation and termination steps
201
ribo in euks
particles made of rRNA and protein
found free n cytoplasm bound to ER and outside of nuclear envelope
3 sites A , E and P site
mRNA bound primarily to the small subunit
Large subunit lining up the tRNAs
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tRNA is
an adaptor molecule that links codons with specific aa
linked to aa by ester bond
named for aa attached tRNA Ala
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aminoacyl tRNAs
tRNAs which are attached to an aa
One tRNA bind to codon
aa attached to 3 prime end
charges or activates the tRNA
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tRNA and anticodons
recognizes codons due to complementarity of anticodon
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amino acyl tRNA synthetases
rather than degrade tRNA after used, they get recharged
| 20 diff ones to attach each to correct tRNA
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charging an tRNA
1. Folds into a 3D shape where there is a perfect binding site for ATP and aa 1 binds
COnformational change occurs
Hydrolysis of ATP yields pyrophosphate
AMP is connected to the aa add onto the aa 2
3 tRNA binds o the pocket elicits the cconformational change , releases AMP
4 next aa tRNA release
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specificity of charging tRNA
shape of tRNA
both anticodon and the 3 prime end of the tRNA are needed to specify the correct amino acid
proofread final product to ensure that correct aa is added because there is no other way to no if the aa is right or not
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Translation overview
starts at N and goes towards C of the protein
| mRNA read from 5 prime to 3 prime
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Initiation over view
components of the translational apparatus come together with the mRNA and a tRNA containg the first tRNA Met comes in
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elongation overview
aa are bought to the ribo by tRNAs
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termination overview
stop codon is recognized by release factor and the protein comes apart
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eukaryotic initiation
Start codon usually AUG encodes Met
Need Met tRNA
Need eIF euk IF a dozen
Process uses GTP for regulation
eIFs with GTP bind to the tRNA Met and then bind the small ribosomal unit
resulting complex binds to the 5 prime cap and recognizes the mRNA
Have small subunit floating around free of a L subunit have E, P and A site
eIF 3 and 1 attached
Ready to go S with regulatory initiation factors
Met tRNA binds to eIF 2 bound to GTP
43S complex formation forms
Initiation factors with the GTP
Preinitiation complex
Proteins congregate together
Take both ends of mRNA and hold together
mRNA ready to go will bind to small subunit ready to initiate translation
First steps
Form S complex, mRNA ready to go
Small ribo track down RNA start from 5 prime cap
tRNA mark where it slips into place, the HB form perfectly, difficult to select for AUG codons to eliminate wrong start sites
Sequences on either side that needs to be there for the snap in place defines where it will be KOzak sequece
Once it finds an AUG situated inside broader sequence, locks on
Enzyme which wants to hydrolyze GTP to GDP but does not occur until we have the complex in place
Float an collect a large subunit associated with factor associated with GTP
5 prime and 3 prime end of mRNA fall apart
Displace factors because not needed
Others join on after conformational changes happen
Build the whole ribosome
Initiation factor sitting in A
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Kozak sequence example
ACCAUG G
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chain elongation
aa added in sequence to the polypep chain
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elongation mechanism
start codon in the P site and next at A
tRNAs are floating around bound to EF-Tu GTP
Happens super fast
Ribo grabs tRNA from environment and see if it fits if not let go
requires Ef-Tu and Ef-TS
when it grabs one that fits GTP hydrolyzes and tRNA in locked in there
GTP hydrolysis allows for binding of tRNA to A site
Amino terminus of the new aa exposed to the carboxyl terminus of the previous one , ribo rRNA catalyzes formation of petide bond the bond is transferred to A site tRNA using Peptidyl transferase
Met COO terminus connected to the 3 prime end of 1st tRNA gets attached to amino terminus of second one
Next another EF ( translocase bound to GTP ) uses GTP hydrolysis to move empty RNA in E site
Whole ribo somes moved down and shifted have not changed
Whole process starts over again
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exit tunnel and polypep created
In exit tunnel get formation of basic secondary structure elements before it exits
Secondary structure will assemble in cytoplasm where they start folding
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chaperones
Shield proteins from crowded environment so that they can fold into a structure
Side chains can cluster together in a process of cell folding
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termination
UAA , UAG or UGA are stop codons
recognized by protein release factors
When you reach a stop codon, there are release factors ( proteins) that recognize the codons in mRNA in a specific manner
3 diff stop codon
GTP regulated step
RF with GTP fits perfectly
Hydrolyzed and this triggers the release of everything including the polypeptides
translation terminatied through GTP hydrolysis
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moelcular chaperones
usually facilitate protein folding
bind polypep chains in early stages of prot folding
can prevent proteins from folding into something that doesn't work but is very low energy
can sometimes rescue proteins and fold properly if folding goes awry
can target proteins to degradation machine
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proteins after synthesized
destined to remain in cytosol or sent to mito, chloro, peroxisomes and nuclear interior are synthesized in the cytoplasm
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post translational import
what signals that are used to direct protein to proper compartment happen via
happens after the
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ER system target protein
transferred as translation takes place
| cotranslational import
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signal recognition particle ( SRP)
mediates cotranslational import
contains both protein and RNA
floats around in the cytoplasm
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co translational import
SRP binds to ER signal sequence in the protein
stalls and stops translation
SRP binds to a receptor on the ER membrane and ribo docks on the mem
GTP binds to SRP receptor and SRP
pore opens and the polypeptide is inserted
once polypeptide is in the pore, GTP is hydrolyzed and SRP is released
signal sequence is cleaved by signal peptidase as polypepe elongates and translocates to the ER lumen
complete polypep is released and the pore closes
the MEt is always in the cytosol
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protein diS isomerase
facilitates diS in protein folding
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unfolded protein response
sensor molecules in ER lumen detect misfolded protiens
| activate pathways to enhance production for proteins which are needed for folding and degradation
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ER associated degradation
mechanism recognizes misfolded proteins andn exports to cytosol where theya re degraded by proteosomes
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default for soluble proteins is
secretion unless they have a specific side chain or signal sequence which target them to otehr organelles
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proteins whose final destination is the ER have
KDEL LYs Asp GLu Leu or related sequence
| GOlgi has receptors which collect all the KDEL proteins and delivers them to the ER
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integral mem proteins on ER
are bound by one or more alpha helical transmembrane signals
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TYpe 1 mechanism
N terminus is in the ER
stop transfer sequence which is a hydrophobic sequence identical or somwhat similar to the signal sequence gets stuck in the ER halts translocation process and moves through side opening in translocon
terminal signal peptide is cut off and have n terminus in the lumen
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TYpe 2
start transfer starts the translocation and moves through side opening in translocon to anchor to the membrane ifhad stop locon will have both N and C terminus in cytoplasm
C terminus is in the ER
internal start transfer sequence
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fate of ER trans membrane
remain in the ER or exported to other mem compartments within the cell
membrane budding and fusion is used to transfer the mem to the appropriate compartment
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FRAP vs FLIP
flourescence recovery after photobleaching
If you point a laser at one part of cell
Bleach all the fllourescent molecules at one spot
If your hypothesis is that they do not move, hole is there
If your hypothesis is that it is fluid, the phospholipids will diffuse into the space
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labelling surface molecules
You can make chemical phospholipids flourescents and put in to the cell
Immunoflourescence typically do on dead cell
- if on surface protein can use antibodies to stain the cell
Don’t need to kill it
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mem fluidity
mouse bilayer one side and human one on other side
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FLIP
flourescence lost in photobleaching
f you want to know which areas providing the phospholipids
Any molecules that are going into the bleached time
If there are any area goes dim
Ring around cell - boum of proteins, half will go dim half will stay
Tells you where the phospholipids are coming from fusing to spot you are bleaching
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GFP
Special forms of flourescent proteins that when activated with light will become flourescent
Activate it and look to see where it goes
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phospholipids include
Glycerol based phosphoglycerides and sphingosine
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fatty acids
Chains of CH2 which vary in length anywhere between 12 to 20 carbonds
Longer they are, more tightly associated they are
dictates usual thickness of the mem
